Recombinant oligomeric protein complexes with enhanced immunogenic potential

ABSTRACT

The present invention relates to a chimeric protein comprising an antigen and an oligomerisation domain. The present invention relates further to recombinant oligomeric protein complexes comprising said chimeric protein and the use thereof for the manufacture of a vaccine.

[0001] The present invention relates to a chimeric protein comprising an antigen and an oligomerisation domain. The present invention relates further to recombinant oligomeric protein complexes comprising said chimeric protein and the use thereof for the manufacture of a vaccine. Many of the natural occurring antigens, irrespective of their origin, are oligomeric in nature. Some non-limiting examples are Hsp16.3 of Mycobacterium tuberculosis and several bacterial toxins. Hsp16.3, an immunodominant antigen of Mycobacterium tuberculosis with serodiagnostic value, occurs as an oligomeric structure, presumably based on trimers (Chang et al, 1996). Cholera toxin as well as the closely related heat-labile toxin from Escherichia coli is composed of two subunits, A and B, which form an oligomeric assembly AB₅. The B subunit portion of cholera toxin can be used as an immunizing agent in humans, providing protection against both cholera and diarrhoea caused by enterotoxigenic E. coli (Sanchez et al. 1990). In a similar way, B oligomer, which is a constituent of pertussis toxin, can be used to elicit immunoprotective responses against Bordetella pertussis.

[0002] Also viral antigens occur often as oligomeric structures. As non-limiting examples, we can cite the herpes simplex virus type 1 glycoprotein B (Lin et al., 1996) or the flavivirus non-structural protein NS1, which has been used in a recombinant vaccine against dengue-2 and which exists normally as a homodimer in infected cells (Winkler et al., 1988). The HIV-1 envelope molecules gp4l and gp120, which may be interesting targets for vaccine development, likewise form an oligomeric structure (Burton, 1997). The predominant antigens of influenza virus are also oligomers. Haemagglutinin (HA) occurs naturally as a homotrimer. Neuraminidase (NA) occurs naturally as a homotetramer, composed of two disulphide-linked dimers, which are held together by non-covalent interactions (Laver and Valentine, 1969; Bucher and Kilbourne, 1972; Varghese et al., 1983; Ward et al., 1983). Influenza M2-protein also occurs normally as a homotetramer.

[0003] Most of the vaccines on the market are either inactivated or attenuated life vaccines. These vaccines are often produced in animal cell culture, implying a high level of biohazard because of the direct handling of pathogenic viruses, and with a high production cost due to expensive raw materials and complicated product processes.

[0004] Although the oligomeric antigens are presumed to keep their oligomeric structure in these vaccines, this is not always the case. For example in the preparation of influenza split vaccines, the oligomeric antigens may lose their oligomeric structure during the viral disruption step. For influenza viruses, there is an additional complication due to the fact that they undergo significant antigenic variation in their two surface antigens haemagglutinin (HA) and neuraminidase (NA) (antigenic drift). Due to the variability of these two proteins a broad spectrum, long lasting vaccine against influenza has so far not been developed. The influenza vaccine commonly used has to be adapted almost every year to follow the antigenic drift. When more drastic changes occur in the virus, known as antigenic shift, a previous vaccine is no longer protective. The present vaccines are based on virus material that has been produced in chicken eggs. Although these vaccine preparations have been found to be effective against an influenza infection caused by the homologous virus strain, there are several drawbacks, such as the production time, which makes it impossible to put an adapted vaccine on the market on short term notice. Moreover, considering the antigenic drift or even the sudden appearance of a shift variant, there is the added risk of selecting antigenic variants of the virus by the growth process in the eggs itself (Koduhalli et al., 1995).

[0005] Therefore, considerable effort has been put in the development of recombinant vaccines. Recombinant vaccines can be based on selected epitopes and are generally safer and cheaper to produce. Moreover, as described e.g. in WO9319185, in recombinant vaccines, the antigen can be fused to immunostimulatory domains to avoid or limit the use of adjuvant in the vaccine preparation, or to boost the immune response of weakly immunogenic epitopes. Recombinant vaccines have been developed against several major diseases, such as measles, tetanus, pertussis, TBC, hepatitis B, cholera and influenza. Several others, such as e.g. a recombinant vaccine against HIV, are under development.

[0006] As an example, recombinant vaccines against influenza virus have been described in WO9406468, WO9407533, WO9518861 and WO9520660. Moreover, a recombinant vaccine based on the membrane protein M2 has been developed, which has the additional advantage to induce a broad spectrum, long-term protection (Neirynck et al., 1999; WO9303173, WO9907839, WO9928478).

[0007] Both NA (Laver and Valentine, 1969; Bucher and Kilboume, 1972; Varghese et al., 1983; Ward et al., 1983) and M2 (Sugrue and Hay, 1991) occur naturally as homotetramers. However, recombinant preparations of NA only partly yield homotetramers, the major part of the preparation being monomers and dimers.

[0008] Although one might expect that antigenic epitopes are as well, if not better accessible in these mono- and dimers, surprisingly, we found that tetrameric molecules were considerably superior in eliciting a specific antibody response. Although tetrameric preparations of these molecules can be obtained by an additional purification step of the recombinant protein, such as size exclusion chromatography, such purification would lead to an important loss in yield. Furthermore, renewed dissociation of the purified material cannot be excluded. By fusing an oligomerisation domain to the influenza antigen, a spontaneous tetramerisation could be obtained of the fusion protein, yielding a recombinant protein in an almost purely tetrameric form. Surprisingly, we found that these fusion tetramers showed a similar enhanced antigenic capacity as the purified tetrameric, recombinant protein.

[0009] It is the object of the invention to provide a highly immunogenic recombinant antigen protein complex, preferably a recombinant influenza antigen protein complex.

[0010] It is a first aspect of the invention to provide a chimeric protein comprising antigen, derived from a naturally occurring oligomeric protein complex, and an oligomerisation domain. Said oligomerisation domain is heterologous, i.e. it is derived from another protein than the antigen. The oligomerisation domain is driving the oligomerisation of the chimeric protein to form a recombinant oligomeric protein complex. Preferentially, the degree of oligomerisation (dimeric, trimeric, tetrameric or higher) of the chimeric protein is identical to the degree of oligomerisation of the naturally occurring protein complex from which the antigen is derived and the antigenic domain of the chimeric protein is presented in a tertiary structure that is similar or identical to that of the tertiary structure of the antigenic domain in the naturally occurring protein. Preferably, said antigen is an influenza antigen. More preferably, said antigen is influenza neuraminidase or influenza M2-protein or a functional fragment thereof. Most preferably, said antigen is chosen from the group consisting of influenza A neuraminidase, influenza A M2 or influenza B NB-protein. Oligomerisation domains are known to the people skilled in the art and have, amongst others, been described in WO9637621, WO9818943, WO9856906, WO9962953 and WO0069907. Preferentially, the oligomerisation domain is a leucine zipper. More preferentially, the oligomerisation domain is a leucine zipper derived from the yeast transcription factor GCN4, or a modified form thereof. Most preferentially, the oligomerisation domain is a modified leucine zipper, derived from the yeast transcription factor GCN4, as described by Harbury et al. (1993). A preferred embodiment of the invention is the influenza B neuraminidase or a functional fragment thereof such as the neuraminidase B ecto-domain, fused to an oligomerisation domain such as a modified leucine zipper, derived from the yeast transcription factor GCN4, as described by Harbury et al. (1993). Another preferred embodiment is the influenza B NB-protein, or a functional fragment thereof, fused to an oligomerisation domain.

[0011] It is an aim of the present invention to present the recombinant protein complex in a conformation that resembles the conformation of the naturally occurring protein complex. In cases where the naturally occurring protein complex has a defined enzymatic activity, as is the case for influenza neuraminidase, the recombinant protein complex will have a comparable enzymatic activity.

[0012] Apart for the oligomerisation domain and the antigen, the chimeric protein may comprise other polypeptide sequences, such as linker sequences or polypeptide sequences that enhance the immune response. Such polypeptide sequences that enhance the immune response are known to the person, skilled in the art and include, as a non-limiting example, one or more copies of the third complement protein fragment d (C3d; Dempsey et al., 1996) or tetanus toxin fragment C, or Escherichia coli enterotoxin fragment A or B, or T-cell epitopes derived from the same pathogen as the antigen.

[0013] Another aspect of the invention is a recombinant oligomeric protein complex comprising a chimeric protein according to the invention. Preferentially, said recombinant oligomeric protein complex is a dimer, or a tetramer. More preferentially, said recombinant oligomeric protein complex is a homodimer or a homotetramer. Preferably, said oligomeric protein complex elicits a higher immune response than the monomeric subunit. A preferred embodiment of the invention is a recombinant oligomeric protein complex that has a comparable enzymatic activity as the naturally occurring oligomeric protein complex of which the antigen, comprised in the chimeric protein that forms said recombinant oligomeric protein complex, is derived.

[0014] Still another aspect of the invention is a nucleic acid, encoding a chimeric protein according to the invention. A preferred embodiment is a nucleic acid, comprising the sequence presented in SEQ ID N° 1. Another preferred embodiment is a nucleic acid, comprising the sequence presented in SEQ ID N° 3. Still another preferred embodiment is a nucleic acid comprising the sequence presented in SEQ ID N° 5.

[0015] A special embodiment is a nucleic acid, according to the invention that may be used in DNA vaccination. Vectors for DNA vaccination are known to the person skilled in the art and are described, amongst others, in WO9604394, WO9728259 and WO9908713. Methods for DNA vaccination have been described amongst others in WO0012121.

[0016] A further aspect of the invention is an expression vector comprising a nucleic acid according to the invention and allowing the expression of a chimeric protein, according to the invention. Said expression vector can be any eukaryotic or prokaryotic expression vector, as known to the person skilled in the art. In one preferred embodiment, the expression vector is pACGCN4NAs (Deposition at BCCM—(deposit nr LMBP 4270)), comprising the antigenic head domain of NA wherein the N-terminal part has been replaced by a modified leucine zipper derived from the yeast transcription factor GCN4, which is imposing tetramerisation (Harbury et al., 1993). This construct is fused to the secretion signal of influenza haemagglutinin and the construct is placed under control of the baculovirus polyhedrin promoter. Another preferred embodiment is pACsM2eGCN4 (deposit nr LMBP 4271), comprising the ectodomain of M2, fused at its N-terminus to the GP67 secretion signal of Baculovirus and at its C-terminus to the tetramerising, modified leucine zipper derived from the yeast transcription factor GCN4 (Harbury et al., 1993). Still another preferred embodiment is pACsM2eGCN4C3d (Deposition at BCCM—(deposit nr LMBP 4463)), where said ectodomain of M2, placed after the GP67 secretion signal of Baculovirus and fused to the GCN4 leucine zipper, is fused to the C3d domain.

[0017] Still another aspect of the invention is a host cell, transformed with an expression vector according to the invention. Said cell can be any prokaryotic or eukaryotic host cell. Transformation procedures are known to the person skilled in the art.

[0018] A further aspect of the invention is the use of a chimeric protein according to the invention and/or the use of a recombinant oligomeric protein complex according to the invention for the preparation of a vaccine against influenza. A special embodiment is the use of said chimeric protein whereby said chimeric protein comprises SEQ ID N° 2, SEQ ID N° 4 or SEQ ID N° 6. The chimeric protein may also be used to elicit monoclonal or polyclonal antibodies, using techniques known to the person skilled in the art, whereby said antibodies can be used for diagnostic or therapeutic purposes. Human or humanised antibodies may be especially useful as therapeutics for people with a decreased immune response, such as HIV patients.

[0019] Still another aspect of the invention is a vaccine against influenza, comprising a chimeric protein and/or a recombinant oligomeric protein complex according to the invention.

[0020] Still another aspect of the invention is a vaccine against influenza, comprising a substantially pure tetrameric form of a recombinant influenza antigen. In one preferred embodiment, said recombinant influenza antigen is recombinant influenza neuraminidase, or a functional fragment thereof. In another preferred embodiment, said recombinant influenza antigen is recombinant M2, or a functional fragment thereof.

[0021] More preferably, said influenza vaccine is fused to a heterologous oligomerisation domain, most preferably, said influenza vaccine is fused to the tetramerising, modified leucine zipper derived from the yeast transcription factor GCN4 (Harbury et al., 1993). A preferred embodiment is a vaccine, comprising a substantially pure tetrameric form of neuraminidase or a functional fragment thereof, whereby said substantially pure tetrameric form has an enzymatic activity that is comparable to naturally occurring influenza neuraminidase.

[0022] Still another aspect of the invention is the use of chimeric protein according to the invention and/or the use of a recombinant oligomeric protein complex according to the invention to screen inhibitors of the biological activity of the naturally occurring protein complex. A preferred embodiment is said use to screen inhibitors of influenza A or influenza B neuraminidase, or inhibitors of influenza A M2 or influenza B NB-protein.

DEFINITIONS

[0023] Chimeric protein as used here means that the protein is composed of at least two polypeptides, which do not occur in the same protein in the natural form.

[0024] Antigen as used here means an antigen, derived from a naturally occurring oligomeric protein from a pathogenic organism or micro-organism, including viruses, and can be used in a host, preferably, but not limited to a human host, to elicit an immune response against said pathogenic organism or micro-organism.

[0025] Recombinant oligomeric protein complex is a protein complex wherein at least one of the subunits is a chimeric protein comprising an oligomerisation domain.

[0026] Recombinant antigen as used here means that said antigen is produced by recombinant DNA techniques. The antigen may be produced in any prokaryotic or eukaryotic host cell, such as, as a non-limiting example, Escherichia coli, Bacillus subtilis, yeast such as Saccharomyces cerevisiae or Kluyveromyces sp., fungal cells, insect cells, plant cells or mammalian cells.

[0027] Comparable enzymatic activity means that both the naturally occurring protein complex and the recombinant oligomeric protein complex can perform at least one identical biochemical transformation of at least one substrate, but that the specific activity of the enzymatic complexes may differ. Identical biochemical transformation means that the substrate by the action of the enzymatic activity is transformed in an identical end product or identical end products.

[0028] Substantially pure tetrameric form of a recombinant antigen means that said recombinant antigen is at least for 80%, preferably at least for 90% in the tetrameric form.

[0029] Functional fragment of influenza neuraminidase or of influenza M2 is any fragment that can elicit an immune response against influenza virus. As a non-limiting example, a functional fragment of influenza M2 is the M2 ecto-domain.

[0030] Unless it is explicitly mentioned as DNA vaccine, vaccine as used here can be any protein based vaccine, including injectable as well as mucosal vaccines.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1: Construction of pACGCN4NAs

[0032] Phprom: polyhedrin promoter

[0033] sHA: secretion signal of the influenza haemagglutinin

[0034] GCN4: modified GCN4 leucine zipper

[0035] NA: neuraminidase

[0036] bla: β-lactamase

[0037] ori: origin of replication

[0038] bold grey line: baculovirus homology region

[0039]FIG. 2: Construction of pACsM2eGCN4

[0040] Phprom: polyhedrin promoter

[0041] sGP67: secretion signal of the baculovirus GP67 protein

[0042] M2e: extracellular part of M2 (M2 ectodomain)

[0043] GCN4: modified GCN4 leucine zipper

[0044] bla: β-lactamase

[0045] ori: origin of replication

[0046] bold grey line: baculovirus homology region

[0047]FIG. 3: Flow diagram for the construction of pACsM2eGCN4C3d. Only restriction sites relevant to the cloning procedure have been indicated.

[0048]FIG. 4: Antibody response against different oligomeric subforms of recombinant neuraminidase.

[0049]FIG. 5: Survival of mice vaccinated with different oligomeric subforms of recombinant neuraminidase after a lethal challenge with homologous, mouse-adapted influenza virus.

[0050]FIG. 6: Analysis of GCN4NAs expression by Western blot.

[0051]FIG. 7: Sucrose gradient pattern of secreted neuraminidase and secreted chimeric GCN4NAs.

[0052]FIG. 8: Proposed structure of GCN4NAs based on the known structure of its two constituent domains.

[0053]FIG. 9: GCN4NAs is a tetramer as shown by chemical cross-linking of its subunits.

[0054] Lane 1: 30 min incubation of GCN4NAs with 1.2 mM BS3

[0055] Lane 2: 5 min incubation of GCN4NAs with 1.2 mM BS3

[0056] Lane 3: 30 min incubation of GCN4NAs with 0.6 mM BS3

[0057] Lane 4: 5 min incubation of GCN4NAs with 0.6 mM BS3

[0058] Lane 5: 30 min incubation of GCN4NAs with 0.3 mM BS3

[0059] Lane 6: 5 min incubation of GCN4NAs with 0.3 mM BS3

[0060] Lane 7: GCN4NAs without cross-linker

[0061] Lane 8: MW

[0062]FIG. 10: Characterisation of sM2eGCN4 expression.

[0063]FIG. 11: Analysis by 12% SDS-PAGE of proteins secreted by baculovirus-infected Sf9 cells in TC100 medium. TCA precipitated proteins from 800μl medium were dissolved in loading buffer. Lanes A1 and B1 were loaded with proteins derived from cells infected with baculovirus obtained by recombination with an empty expression vector. Lanes A2 and B2 were loaded with proteins derived from cells infected with baculovirus containing the genetic information for expression of sM2eGCN4C3d.

[0064] A: Detection by Western blot analysis using the monoclonal antibody 2C9 (Neirynck et al., 1999).

[0065] B: Proteins were stained with SyproOrange (Molecular Probes, Eugene, Oreg. USA). An arrow indicates the position of the recombinant protein sM2eGCN4C3d.

[0066] M1=‘Benchmark’ prestained protein molecular weight markers (Gibco BRL, Bethesda, Md., USA). M2 =protein molecular weight markers from Molecular Probes (Eugene, Oreg., USA)

[0067]FIG. 12: Gel filtration chromatography on a Superdex 200 HR column (Amersham Pharmacia Biotech, Uppsala, Sweden).

[0068] (A) Elution profile after separation of a mixture of reference proteins for calibration.

[0069] (B) Elution profile of partially purified, recombinant sM2eGCN4C3d; fractions were analyzed by Western blot as described in the legend to FIG. 11.

[0070]FIG. 13: sM2eGCN4C3d is a tetramer as shown by chemical cross-linking of its subunits.

[0071] Western blot analysis of sM2eGCN4C3d before (lane 1) and after treatment with 4, 6 and 12 mM cross-linking agent BS3 (lanes 2, 3 and 4, respectively). Proteins were denatured in Laemmli buffer in the presence of DTT as reducing agent, and separated on a 5-14% gradient SDS-polyacrylamide gel. Blotting of the proteins was followed by screening with anti-M2 monoclonal antibody 2C9, followed by secondary antibody (rat anti-mouse IgG-peroxidase conjugate; Sigma Chemical Company, St. Louis, Mo., USA). Positive signals were revealed after addition of “Renaissance” chemiluminescent substrate solution (NEN Life Science Products, Boston, Mass, USA).

[0072]FIG. 14: Antibody response against sM2eGCN4C3d

[0073] Mice were injected i.p. either with PBS +adjuvant or sM2eGCN4C3d +adjuvant, and the injections were repeated twice at 2 weeks interval (cf. Example 13). Serum was taken 10 days after each injection, and antibody titers were determined using either M2e-peptide or sM2eGCN4C3d for trapping. Vac, b1 and b2: serum taken after first injection, first boost and second boost, respectively.

[0074]FIG. 15. Survival of mice after a challenge with mouse-adapted influenza virus

[0075] Balb/c mice were immunized three times with PBS or 10 μg sM2eGCN4C3d (cf. Example 13), and challenged with homologous, mouse-adapted X47 virus

EXAMPLES Example 1 Construction fpACGCN4NAs

[0076] All PCR amplifications were carried out using Vent polymerase (New England Biolabs, Beverly, Mass., USA), with a total of 10 cycles and 200 ng plasmid for all reactions. The cycling conditions were as specified for the individual amplifications. The oligonucleotides used for the constructions are shown in Table 1.

[0077] The baculovirus transfer vector pAC2IVNAs (containing the cDNA sequence of the N2 neuraminidase of A/Victoria/3/75 influenza virus of which the membrane anchor was substituted by the cleavable signal sequence of the haemagglutinin; described in detail in Deroo et al., 1996) was used as a template to generate two PCR products using the primer pairs BACfor/GCN4nh (denaturation: 94° C., 1 min; annealing 57° C., 1 min; synthesis 75° C., 30 sec) and GCN4cooh/BACrev (denaturation: 94° C., 1 min; annealing 59° C., ₁ min; synthesis 75° C., 2 min 30 sec), which were subsequently digested with PstI/HindIII and BcII/Xbal; respectively. The resulting fragments and a 5′end kinated synthetic DNA fragment (GCN4pos/GCN4neg complementary oligonucleotide pair) were then ligated with the PstI/Xbal 10089 bp vector fragment of pAC2IVNAs. The inserted sequence in the baculovirus transfer vector obtained in this way (pACGCN4NAs), codes for the antigenic and catalytic head domain of the neuraminidase (NA) preceded by a modified GCN4 leucine zipper which promotes the formation of a tetrameric protein (Harbury et al., 1993), and by the secretion signal of the influenza haemagglutinin (sHA). Expression of the fusion gene is controlled by the baculovirus polyhedrin promotor (Phprom). The corresponding recombinant baculovirus (designated AcNPV[GCN4NAs]) was generated by calcium phosphate cotransfection of Sf9 insect cells with BaculoGold baculovirus DNA (Pharmingen, San Diego, Calif., USA), following the procedure as described in King and Possee (1992). The construction is summarized in FIG. 1.

Example 2 Construction of pACsM2eGCN4

[0078] After PCR amplification of the baculovirus GP67 secretion signal (primers GP67s and GP67a; denaturation: 94° C., 1 min; annealing 62° C, 1 min; synthesis 75° C., 45 sec) out of pACGP67A (baculovirus transfer vector purchased from Pharmingen, San Diego, Calif., USA) and digestion with SpeI/HindIII, the fragment was subcloned in the SpeI/HindIII vector sequence of pUCC3d (described in example 3), resulting in pUCsgp. Following digestion of pUCsgp with HindIII/NaeI, the GP67 secretion signal was fused with the M2e fragment (coding for the ectodomain of the M2 protein) obtained by PCR amplification (primers M2Ss and UM2ECa; denaturation: 94° C., 1 min; annealing: 62° C., ₁ min; synthesis: 75° C., 20 sec) and subsequent treatment with HindIII. This construct, referred to as pUCsgpM2e, was used as a template to generate a new PCR fragment (primers GP67s and M2rev; denaturation: 94° C., 1 min; annealing 56° C., 1 min; synthesis 75° C., 30 sec), which was digested with SpeI and BspEI. The GCN4 PCR fragment obtained from pACGCN4NAs (primers GCNfor and GCNrev; denaturation: 94° C., 1 min; annealing 58° C., 1 min; synthesis 75° C., 30 sec) was then ligated with the former fragment together with the SpeI/BgIII 9610 bp vector fragment of pACGP67A. In the resulting baculovirus transfer vector pACsM2eGCN4, the sequence coding for the M2 ectodomain (M2e) is fused at its C-terminus to a modified GCN4 leucine zipper which is able to induce tetramerization (Harbury et al., 1993), and is preceded by the GP67 secretion signal (sGP67), and by the polyhedrin promoter (Phprom). The corresponding recombinant baculovirus (designated AcNPV[sM2eGCN4]) was generated by calcium phosphate cotransfection of Sf9 insect cells with BaculoGold baculovirus DNA (Pharmingen, San Diego, Calif., USA), following the procedure as described in King and Possee (1992). The construction is summarized in FIG. 2.

Example 3 Construction of pACsM2eGCN4C3d

[0079] The plasmid pSG5.C3d3.YL (Dempsey et al, 1996) was a kind gift from Dr. D.T. Fearon, Department of Medicine, University of Cambridge School of Medicine, Cambridge, UK. The C3d genetic information was first amplified and subcloned in the vector pUC18 (Norrander et al.,1983) as follows: The C3d sequence was PCR amplified (primers C3ds and C3da, pSG5.C3d3.YL as template; denaturation: 94 ° C., 30 sec; annealing: 60° C., 20 sec; synthesis: 72° C., 2 min), followed by cleavage with SpeI and BgIII to allow insertion into SpeI/BgIII digested, intermediate vector pUC18f. The latter had been created by inserting a SpeI restriction site in SmaI opened pUC18, using the adaptor 5′TCACTAGTGA3′, and by inserting a BgIII restriction site in the HindIII opened vector, using the adaptor 5′CAGATCTG3′. The result of cloning the PCR fragment in pUC18f was the plasmid pUCC3d. The starting plasmid pSG5.C3d3.YL was cleaved also with XbaI, followed by blunting with T4 DNA polymerase, and by subsequent digestion with BspEI, which provided a 680 bp fragment. The latter was inserted in the vector pUCC3d previously cleaved with SpeI, blunted with T4 DNA polymerase, and then cut with BspEI. The result was the plasmid pUCC3dEEF. Starting from the plasmid pACsM2eGCN4 (example 2) and using the primers GP67s and GCNrev2, the sM2eGCN4 coding sequence was PCR amplified (denaturation: 94° C., 1 min; annealing: 57° C, 1 min; synthesis: 75° C, 30 sec), and then treated with SpeI and BamHI. The resulting fragment was inserted into SpeI/BamHI digested pACGP67A (baculovirus transfer vector purchased from Pharmingen, San Diego, Calif., USA). This provided the intermediate construct pACsM2eGCN4f. The aforementioned plasmid pUCC3dEEF was digested with BgIII and EcoRI to provide a 1094 bp fragment coding for C3dEEF, which was subcloned in BamHI and EcoRI opened pACsM2eGCN4f. This provided the plasmid pACsM2eGCN4C3d. In this baculovirus transfer vector, the polyhedrin promotor (Phprom) is followed by a multi-component fusion gene coding for the GP67 secretion signal (sGP67), the M2e ectodomain (M2e), the GCN4 leucine zipper, the mouse C3d domain and a C-terminal ‘EEF’ tag. The corresponding baculovirus (designated AcNPV[sM2eGCN4C3d]) was generated by calcium phosphate cotransfection of Sf9 insect cells with BaculoGold baculovirus DNA (Pharmingen, San Diego, Calif., USA), following the procedure as described in King and Possee (1992). The construction is summarized in FIG. 3.

Example 4 Analysis of Antibody Response Against Different Oligomeric Subforms of Purified Secreted Neuraminidase

[0080] Secreted recombinant NA (NAs) was produced by a baculovirus expression system and subsequently purified according to Deroo et al. (1996). After Superdex 200 gel filtration, which is the final step of the purification procedure described in Deroo et al. (1996), the fractions corresponding to monomeric, dimeric and tetrameric NAs were pooled separately, and concentrated by ultrafiltration using Centriplus devices (10 kDa cut-off)(Amicon, Danvers, Mass., USA). Groups of 12 female Balb/c mice (SCK Mol, Belgium) were then immunized three times by subcutaneous injection with 1 μg of a specific oligomeric subform in the presence of a low-reactogenic adjuvant, as described in Deroo et al. (1996). Two weeks after each immunization, blood was collected from the tail vein and serum was prepared. Serum samples were subsequently analyzed by ELISA. For this purpose, 96-well plates were coated with purified, pronase-cleaved NA (Deroo et al., 1993), and incubated with 2-fold serum dilutions. Serum antibody titers were measured by adding alkaline phosphatase conjugated goat anti-[mouse IgG] antibody (Sigma Chemical Co., St. Louis, Mo., USA) and p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo., USA) as a substrate. The serum antibody titer corresponds to the log₂ value of the dilution factor which gave an OD of 0.5 above the control. Mice immunized with tetrameric NAs show a superior antibody respons throughout the immunization regimen (FIG. 4). After 2 immunizations, a ˜17-fold difference was detected in favor of the mice immunized with tetrameric NAs as compared to those that received an equal dose of dimeric NAs, and a ˜137-fold difference compared to the group immunized with an equal dose of monomeric NAs. The difference between tetrameric and monomeric vaccines increased even further after the third immunization, up to a factor of ˜360.

Example 5 Survival of Mice Challenged With a Lethal Dose of Influenza Virus After Vaccination With Different Oligomeric Subforms of Purified, Secreted Neuraminidase

[0081] Groups of 12 female Balb/c mice were immunized subcutaneously with different oligomeric subforms of purified, secreted neuraminidase (NAs), as outlined under example 4. Three weeks after the third injection, mice were challenged with a potentially lethal dose of mouse-adapted influenza virus (X47 reassortant strain) as described in Deroo et al. (1996). No lethality was observed among the animals that received tetrameric NAs vaccine. By contrast, only 75% and 50% of the animals immunized with dimeric and monomeric NAs, respectively, survived the challenge (FIG. 5).

Example 6 Analysis of GCN4NAs Expression by Western Blotting

[0082] Log-phase Sf9 insect cells were inoculated with the indicated baculovirus at high multiplicity of infection (>10). Cells were subsequently transferred to serum-free TC100 medium (Gibco BRL, Bethesda, Md., USA) and further incubated for 48 h before harvesting the supernatant. Proteins were precipitated by adding an equal volume of acetone (pre-equilibrated at −20° C.) and subsequently analysed by 12% reducing SDS-PAGE followed by Western blotting. Bands were visualized using a rabbit polyclonal IgG antibody against pronase-cleaved neuraminidase, followed by incubation with a secondary antibody (goat anti-[rabbit IgG]—alkaline phosphatase conjugate; Sigma Chemical Co., St. Louis, Mo., USA) and BCIP/NBT substrate solution. Secreted GCN4-fused neuraminidase (GCN4NAs), after denaturation, is clearly visible as a band of˜55 kDa (FIG. 6).

Example 7 Sucros Gradient C ntrifugation of NAs V rsus GCN4NAs

[0083] Secreted neuraminidase (NAs) and secreted GCN4-fused neuraminidase (GCN4NAs) were produced as outlined under example 4 by infection of Sf9 cells with the recombinant baculovirus described in Deroo et al. (1996) or with AcNPV[GCN4NAs], respectively. Harvested supernatant (10 ml) was concentrated using Centriplus devices (10 kDa cut-off) (Amicon, Danvers, Mass., USA) until ˜1 ml, and subsequently supplemented with 1 volume of 100 mM Tris.Cl pH7.4, 200 mM NaCI, 0.2% Triton X-100, containing a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals, Basel, Switzerland). Alternatively, when working with large volumes of harvested supernatant (200 ml), GCN4NAs was precipitated with ammonium sulfate. Material precipitating between 60% and 80% (NH₄)₂SO₄ saturation, was collected by centrifugation (10000 g, 60 min) and dissolved in 50 mM Tris.Cl pH7.4, 100 mM NaCl, 0.2% Triton X-100 containing a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals, Basel, Switzerland). This solution containing GCN4NAs was dialysed against the same buffer.

[0084] After centrifugation to remove insoluble components (10 min at 14000 rpm), 1 ml sample was loaded on a 30 ml continuous sucrose gradient (5% -25%) made up in 10 mM Tris.Cl pH 7.4, 100 mM NaCl, 0.1% Triton X-100, and supplemented with Complete protease inhibitors. Gradients were centrifuged at 10° C. in a SW28 Beckman rotor for 16 h at 28000 rpm. Fractions of 1.5 ml were then collected from the bottom of the gradient and analysed by ELISA and by enzymatic activity. ELISA was carried out in 96-well plates coated with a rabbit IgG fraction raised against purified, pronase-cleaved neuraminidase (cf. example 6). Gradient fractions were added to the plates, and bound NAs was detected by addition of biotin-conjugated, anti-pronase-cleaved NA rabbit IgG. Plates were developed by incubating them with streptavidin -alkaline phophatase conjugate (Gibco BRL, Bethesda, Md., USA) and p-nitrophenyl phosphate as a substrate. OD values were read at 405 nm. Analysis of enzymatic activity was performed as described in Deroo et al. (1996). The profile obtained with NAs following sucrose gradient centrifugation is in agreement with the gel filtration profile of purified NAs described in Deroo et al. (1996), and typically results in two major peaks corresponding to catalytically active, tetrameric NAs, and catalytically non-active, dimeric NAs, respectively, the latter form representing about ⅔ of the total amount of secreted NAs (FIG. 7). The amount of monomeric NAs was below detection levels in this experimental set-up. By contrast, GCN4NAs was completely secreted as a catalytically active, tetrameric protein, with no detectable amounts of oligomeric forms of lower order. Its sedimentation at the same rate as recombinant, tetrameric NAs indicates a similar molecular mass. The relative specific enzymatic activities of the sucrose gradient purified proteins are compared in Table II. A model of the recombinant tetrameric protein complex is shown in FIG. 8.

Example 8 Characterisation of GCN4NAs by Cross-linking

[0085] Samples of partially purified GCN4NAs obtained by sucrose gradient centrifugation, as describes in example 7, were concentrated using Microcon devices (10 kDa cut-off) (Amicon, Danvers, Mass., USA) and dialysed using Spectra/Por SispoDialyzer (8 kDa cut-off) (Spectrum, Rancho Dominguez, Calif., USA) overnight against PBS to remove Tris buffer. Cross-linkers were obtained from Pierce (Rockford, Ill., USA). Bis(sulfosuccinimidyl)suberate (BS3) was added from a freshly made 20 mM stock solution in DMSO to final concentrations of 2 to 6 mM. The reactions were incubated for 1 hour at room temperature, and quenched for 15 minutes by addition of Tris buffer, pH 8, to a final concentration of 300 mM. After cross-linking, an equal volume of 2 ×SDS loading buffer (5% SDS, 100 mM DTT, 20% glycerol, 5 mM EDTA, 50 mM Tris buffer, pH 8) was added and the sample was boiled for 5 minutes. SDS PAGE analysis was carried out on a MiniProtean II apparatus (BioRad, Hercules, Calif., USA) using 4-15% precast gradient gels. Electroblotting from SDS PAGE gels onto nitrocellulose (NC) membranes was performed using a Mini Trans-Blot cell (BioRad, Hercules, Calif., USA) and required 45 min at 100 V. Thereafter, NC membranes were blocked in PBS containing 2% BSA for 2h. Blots were incubated with anti-NA rabbit serum (cf. example 6) diluted (1/5000) in PBS containing 1% BSA and 0.1% Tween-20 (PBT). After washing away unbound antibodies, alkaline phosphatase conjugated goat anti-rabbit-IgG serum (Organon Teknika, West Chester, Penn., USA) was added at a dilution of 1/7000 in PBT. Detection was achieved with NBT/BCIP (Roche Diagnostics, Indianapolis, Ind., USA). Prestained, broad range molecular weight marker (BioRad, Hercules, Calif., USA) was used for reference. Incubation of GCN4NAs in the presence of the cross-linker BS3 resulted in the formation of covalently linked oligomers, as depicted in FIG. 9. The major crosslinked species have an estimated molecular mass of 250 kDa and 115 kDa, corresponding to cross-linked tetramers and dimmers, respectively. The presence of the dimeric forms decreased with increasing concentration of cross-linker. This result confirms data obtained by sedimentation and by enzymatic activity, which all indicate that GCN4NAs exists in solution as a tetramer.

Example 9 Characterisation of sM2eGCN4 Expression

[0086] Log-phase Sf9 insect cells were inoculated with baculovirus AcNPV[sM2eGCN4] (example 2) or control virus at high multiplicity of infection (>10). Cells were subsequently transferred to serum-free TC100 medium (Gibco BRL, Bethesda, Md., USA) and further incubated for 48 h before harvesting the supernatant. Proteins were precipitated by adding an equal volume of acetone (pre-equilibrated at −20° C), dissolved in loading buffer and separated by 15% reducing SDS-PAGE, followed by Western blotting. Bands were visualized using the monoclonal antibody 2C9 directed against the M2e domain (Neirynck et al. 1999), followed by incubation with a secondary antibody (goat anti-[mouse IgG] -alkaline phosphatase conjugate; Sigma Chemical Co., St. Louis, Mo., USA) and BCIP/NBT substrate solution. Secreted GCN4-fused M2e (sM2eGCN4), after denaturation, was detected as a band of ˜10.5 kDa (FIG. 10).

Example 10 Characterisation of sM2eGCN4C3d Expression

[0087] Log-phase Sf9 insect cells were inoculated with baculovirus AcNPV[sM2eGCN4C3d] (cf. example 3) or control virus (“mock infection”) at high multiplicity of infection (>10). Cells were subsequently transferred to serum-free TC100 medium (Gibco BRL, Bethesda, Md., USA) and further incubated for 48 h before harvesting the supernatant. Proteins were analyzed by precipitation with ice-cooled TCA and subsequently separated by 12% reducing SDS-PAGE, followed by SyproOrange staining (Molecular Probes, Eugene, Oreg., USA). Secreted sM2eGCN4C3d was revealed as a band of˜41 kDa, which was not present in the medium of ‘mock’ infected Sf9 cells (FIG. 11). For ‘mock’ infection, baculovirus has been generated by calcium phosphate cotransfection of Sf9 cells with transfer vector pACGP67A without an insert for expression, and BaculoGold baculovirus DNA (Pharmingen, San Diego, Calif., USA), following the procedure as described in King and Possee (1992).

[0088] By Western blotting, sM2eGCN4C3d was again visualized as a band of˜41 kDa using the anti-M2e monoclonal antibody 2C9 (Neirynck et al., 1999), followed by incubation with a secondary antibody (rat anti-[mouse IgG]—peroxidase conjugate; Sigma Chemical Co., St. Louis, Mo., USA) and ‘Renaissance’ chemiluminescent substrate solution (NEN Life Science Products, Boston, Mass., USA). The same band was detected when rat monoclonal antibody YL ½ against the ‘EEF’ tag (Abcam Ltd, Cambridge, UK) was used, or sheep polyclonal antibody against mouse complement 3 (Biogenesis, Poole, UK). The following secondary antibodies were used: mouse monoclonal anti-rat kappa and lambda light chains (clones RT-39 & RL-6) conjugated with alkaline phosphatase, and donkey anti-sheep IgG conjugated with alkaline phosphatase, respectively (Sigma, St. Louis, Mo., USA).

[0089] For further characterization, the blotted proteins were stained with amido black, the ˜41 kDa band was isolated by cutting the membrane, and N-terminal sequence analysis was carried out according to Bauw et al. (1987). The first four amino acids were identified as serine, leucine, leucine and threonine, respectively. This result demonstrates that cleavage of the GP67 signal sequence has been executed correctly, and confirms that the˜41 kDa band is without doubt sM2eGCN4C3d.

Example 11 Characterization of the Oligomeric Status of sM2eGCN4C3d

[0090] Secreted, recombinant sM2eGCN4C3d was produced by a baculovirus expression system, as described in example 10. The following purification steps, summarized in Table III, were carried out: First, crude medium was fractionated by differential ammonium sulphate precipitation. Ammonium sulphate was added to the medium, cooled on ice, until 45% saturation and precipitated proteins were removed by centrifugation (Sorvall rotor SS34, 1 hour at 15000 rpm, 4° C.). The (NH₄)₂SO₄ concentration was raised to 95% saturation, and the resulting precipitate was collected by centrifugation. The pellet was redisolved in 50 mM Tris.CI buffer, pH 7.2, (approximately {fraction (1/20)}th of the original volume of the medium), and desalted over a HiPrep Sephadex G25 column (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in 50 mM Tris.CI, pH 7.2. The desalted protein solution was loaded on a Q-Sepharose FF column (h: 9 cm, d: 1.5 cm; Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in 50 mM Tris buffer, pH 7.2, and elution was by a salt gradient of 200 to 400 mM NaCI in 20 mM Tris buffer, pH 7.2. A 100 μl aliquot of each fraction was used for analysis: Proteins were precipitated by TCA from 100 μl aliquots from each fractions in preparation for analysis by 12% SDS-PAGE followed by Western blotting and screening with the anti-M2e monoclonal antibody 2C9 (Neirynck et al., 1999), (cf. example 9), using as secondary antibody (rat anti-[mouse IgG] -peroxidase conjugate; Sigma chemical Co., St. Louis, Mo., USA) and ‘Renaissance’ chemiluminescent substrate solution (NEN Life Science Products, Boston, Mass., USA). Positive fractions were pooled, and the solution was concentrated using Vivaspin-30 and Centricon-100 ultrafiltration devices (Millipore Corporation, Bedford, Mass., USA).

[0091] Approximately 99% of the recombinant protein was retained by the Centricon-100 membrane, suggesting that the molecular mass of the sM2eGCN4C3d oligomer was higher than 100 kDa, as expected for a tetramer.

[0092] To verify further the oligomeric status of sM2eGCN4C3d, 150 μl of the concentrated, partially purified recombinant protein was loaded on a Superdex 200 HR gel filtration column (Amersham Pharmacia Biotech, Uppsala, Sweden), equilibrated in PBS, and resolved at a flow rate of 0.4 ml/min. This Superdex 200 column had previously been calibrated using a mixture of highly purified proteins (HMW and LMW calibration kits (Amersham Pharmacia Biotech, Uppsala, Sweden). As shown in FIG. 12, sM2eGCN4C3d was detected only in fractions nearly coinciding with those of aldolase (theoretical molecular mass 179 kDa), as can be expected for tetrameric sM2eGCN4C3d.

Example 12 Characterization of the Oligomeric Status of sM2eGCN4C3d by Cross-linking

[0093] Samples (10 μl) of partially purified sM2eGCN4C3d (approx. 0.5 μg, in PBS; cf. example 11) were incubated with cross-linker BS3 (Pierce, Rockford, Ill., USA) at final concentrations of 4 to 12 mM. The cross-linker was added from a freshly made 40 mM stock solution in DMSO. The reactions were incubated for 10 minutes at room temperature, and quenched for 15 minutes by addition of 1 M Tris buffer, pH 7.5, to a final concentration of 300 mM. After incubation, an equal volume of 2×SDS loading buffer (5% SDS, 100 mM DTT, 20% glycerol, 5 mM EDTA, 50 mM Tris buffer, pH 8) was added and the samples were boiled for 5 minutes. SDS-PAGE analysis was carried out on a MiniProtean II apparatus (BioRad, Hercules, Calif., USA) using 4-15% precast gradient gels. Electroblofting from SDS-PAGE gels onto nitrocellulose (NC) membranes was performed using a Mini Trans-Blot cell (BioRad, Hercules, Calif., USA) and required 1 hour at 100 V. Thereafter, NC membranes were blocked overnight at 4° C. in PBS containing 2% BSA. Blots were incubated with anti-M2e monoclonal antibody 2C9 in TBS-T (50 mM Tris buffer, pH 7.0, 50 mM NaCl, and 0.1% Tween-20). After washing away unbound antibodies, the blot was screened with secondary antibody (rat anti-[mouse IgG] -peroxidase conjugate; Sigma chemical Co., St. Louis, Mo., USA) and positive signals were revealed after addition of ‘Renaissance’ chemiluminescent substrate solution (NEN Life Science Products, Boston, Mass., USA). Prestained broad range molecular weight marker proteins (BioRad, Hercules, Calif., USA) were used as references. As shown in FIG. 13, treatment of sM2eGCN4C3d with the cross-linker BS3 resulted in the formation of covalently linked oligomers. The major cross-linked species has an estimated molecular mass of approximately 164 kDa, while the monomer had a molecular mass of approximately 41 kDa. This result confirms data obtained by ultrafiltration and gelfiltration, which all indicate that sM2eGCN4C3d exists in solution as a tetramer.

Example 13 Analysis of the in vivo Antibody Response Against sM2eGCN4C3d

[0094] Secreted, recombinant sM2eGCN4C3d was produced by a baculovirus expression system and purified, as outlined in examples 10 and 11. Fractions containing recombinant protein were concentrated using Vivaspin-30 and Centricon-100 ultrafiltration devices (Millipore Corporation, Bedford, Mass., USA). In order to bring sM2eGCN4C3d in PBSA buffer (171 mM NaCl, 3.4 mM KCI, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄), the latter buffer was added to the concentrated protein solution (˜200 μl) to bring the volume of the sample to 2 ml, after which the solution was concentrated again (˜10-fold) using Centricon-100. This step was repeated twice. Groups of 7 female Balb/c mice (Charles River Laboratories, Sulzfeld Germany) at the age of 8 weeks were immunized by intraperitoneal injection with 10 μg sM2eGCN4C3d in the presence of Ribi adjuvant (25 μg monophosphoryl lipid A and 25 μg trehalose-6,6-dimycolate; cf. Neirynck et al., 1999) per mouse. Control mice received adjuvant dispersed in phosphate-buffered saline. The animals were housed in a temperature-controlled environment with 12 h light/dark cycles, and received food and water ad libitum. Two booster injections were given at two-week intervals by supplementing 10 μg sM2eGCN4C3d with 25 μg monophosphoryl lipid A and 25μg adjuvant peptide (cf. Neirynck et al., 1999) per mouse. Ten days after each immunization, blood was collected from the tail vein and serum was prepared. Serum samples were subsequently analysed by ELISA. For this purpose, 96-well plates were coated overnight at 37° C. with 50 μl of 2 μg/ml M2e peptide in 50 mM sodium bicarbonate buffer, pH 9.7, and then blocked with 200 μl PBS containing 1% BSA during 1 hour. Alternatively, 96-well plates were coated overnight at 4° C. with 50 μl of 10 μg/ml anti-mouse complement 3 polyclonal antibodies (ab3163, Biogenesis Ltd, Poole, UK) and blocked with 150 μl PBS containing 1% BSA during 1 hour. With the latter coating, capturing of sM2eGCN4C3d from insect cell medium was allowed during 1 hour 30 minutes at room temperature. After washing, a series of ½ dilutions of the different serum samples, starting with a {fraction (1/50)} dilution, were loaded on peptide-or sM2eGCN4C3d protein coated wells. Bound antibodies were detected with a peroxidase-labeled antibody directed against mouse IgG1 and IgG3 (Southern Biotechnology Associates, Inc.), respectively, diluted {fraction (1/6000)} in PBS containing 1% BSA and 0.05% Tween-20. After washing, the microtiter plates were incubated for 20 minutes with 3,3′, 5,5′-tetramethylbenzidine liquid substrate for peroxidase (Sigma, St. Louis, Mo., USA). The reaction was stopped by addition of 1 M H₃PO₄ and the absorbance at 450 nm was measured. To obtain the value for the specific reactivity to M2e, the absorbance obtained for pre-immune serum at a given dilution was substracted from the absorbance of post-vaccination and post-boosting sera of the corresponding dilution. As presented in FIG. 14 A and C, anti-M2e antibodies of isotypes IgG1 and IgG3 were induced in vaccinated and boosted mice. As shown in FIG. 14 B and D, similar profiles of antibody responses were obtained when recombinant sM2eGCN4C3d proteins were screened with antisera from vaccinated and boosted mice. Hence, it can be concluded that sM2eGCN4C3d can efficiently induce an antigenic antibody response in mice.

Example 14 Protection of Mice Challenged with a Lethal Dose of Influenza Virus After Vaccination with sM2eGCN4C3d

[0095] Groups of 7 female pathogen-free Balb/c mice (Charles River Laboratories Sulzfeld, Germany) were immunized intraperitoneal with sM2eGCN4C3d, as described in example 13. Two weeks after the last immunisation, the mice were challenged intranasally with 14 LD₅₀ of mouse-adapted (m.a.) X47 (Neirynck et al., 1999). As presented in FIG. 15, sM2eGCN4C3d vaccinees were protected against a lethal dose of homologous m.a. influenza A virus. TABLE I PCR Primers and c mplem ntary oligonucle tid pair PCR primers BACfor: 5′ TTTACTGTTTTCGTAACAGTTTTG 3′ GCN4nh: 5′ TACAGAAGCTTGTCTTCGATTTGTTTCATACCGCCAAGGTCTTGGGC GAAAACC 3′ GCN4cooh: 5′ ATCTGATCAAGAAACTGCTGGGCGAAGGTGGCAAAGAGATATGCCCC AAATTAG 3′ BACrev: 5′ CATTTTATGTTTCAGGTTCAGGG 3′ GP67s: 5′ GCTACTAGTAAATCAGTCACACCAA 3′ GP67a: 5′ CGAAGCTTGCCGGCAAAGGCAGAATGCGCCGCC 3′ M2rev: 5′ ACCATTCCGGATGAATCGTTGCATCTGCAC 3′ M2Ss: 5′ TCTCTGCTGACCGAAGTTGAAAC 3′ UM2ECa: 5′ CGAAGCTTACTAGTTCACGGATCCCCACTTGAATCGTTGCATCTGCA CCC GCN4for: 5′ AGATTTCCGGAGGTATGAAACAAATCGAAGAC 3′ GCN4rev: 5′ ATAGGAGATCTATTCGCCCAGCAGTTTCTTG 3′ GCNrev2: 5′ TATTGGATCCGGTGAACCTGATCCTTCGCCCAGCAGTTTCTTG 3′ C3ds: 5′ CCGCGCCCACCCGACGAGATCTCGGATCTACCCCC 3′ C3da: 5′ GCACTAGTTCAAGGATCCGATCCGAACTCTTCAGATCC 3′ c mplementary oligonucle tid pair GCN4pos: 5′ AGCTGGAAGAAATCCTTTCGAAACTGTACCACATCGAAAACGAGCTG GCCAG 3′ GCN4neg: 5′ GATCCTGGCCAGCTCGTTTTCGATGTGGTACAGTTTCGAAAGGATTT CTTCC 3′

[0096] TABLE II Estimated relative values of the specific activities of (tetrameric) NAs versus GCN4NAs tetrameric dimeric monomeric NAs 100% non-active non-active GCN4NAs >70%

[0097] The peak values of the enzymatic activity and the ELISA read-out obtained after sucrose gradient centrifugation were used to deduce a rough estimation of the relative specific activities. The specific enzymatic activity of GCN4NAs amounts to at least 70% of that of the tetrameric form of the unfused NAs protein. TABLE III Purification of sM2eGCN4C3d Volume Protein sM2eGCN4C3d Yield Purification Steps (ml) (mg) (mg) (%) (-fold) Crude 1200 180 2.4 100 1.0 medium 45-95% 48 58 1.9 79 2.5 (NH₄)₂SO₄ precipitate Sepharose 30 6.5 0.9 37 11 Q Superdex 6 1.2 0.6 17 38 200

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1 22 1 1350 DNA Artificial Sequence part of pACGCN4Nas 1 atg aag act atc att gct ttg agc tac att ttc tgt ctg gtt ttc gcc 48 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 caa gac ctt ggc ggt atg aaa caa atc gaa gac aag ctg gaa gaa atc 96 Gln Asp Leu Gly Gly Met Lys Gln Ile Glu Asp Lys Leu Glu Glu Ile 20 25 30 ctt tcg aaa ctg tac cac atc gaa aac gag ctg gcc agg atc aag aaa 144 Leu Ser Lys Leu Tyr His Ile Glu Asn Glu Leu Ala Arg Ile Lys Lys 35 40 45 ctg ctg ggc gaa ggt ggc aaa gag ata tgc ccc aaa tta gtg gaa tac 192 Leu Leu Gly Glu Gly Gly Lys Glu Ile Cys Pro Lys Leu Val Glu Tyr 50 55 60 agg aat tgg tca aag cca caa tgt aaa att aca gga ttt gca cct ttc 240 Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly Phe Ala Pro Phe 65 70 75 80 tct aag gac aat tca att cgg ctt tct gct ggt ggg gac att tgg gtg 288 Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val 85 90 95 acg aga gaa cct tat gtg tca tgc gat cct ggc aaa tgt tat caa ttt 336 Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys Cys Tyr Gln Phe 100 105 110 gca ctc ggg cag ggg acc aca cta gaa aac aaa cat tca aat gac aca 384 Ala Leu Gly Gln Gly Thr Thr Leu Glu Asn Lys His Ser Asn Asp Thr 115 120 125 ata cat gat aga acc cct cat cga acc cta ttg atg aat gag ttg ggt 432 Ile His Asp Arg Thr Pro His Arg Thr Leu Leu Met Asn Glu Leu Gly 130 135 140 gtt cca ttt cac ttg gga acc agg caa gtg tgt ata gca tgg tcc agc 480 Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile Ala Trp Ser Ser 145 150 155 160 tca agt tgt cac gat gga aaa gca tgg ctg cat gtt tgt gtc act ggg 528 Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly 165 170 175 tat gat aaa aat gca act gct agc ttc att tac gat ggg agg ctt gta 576 Tyr Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val 180 185 190 gac agc att ggt tca tgg tct caa aat atc ctc agg acc cag gag tcg 624 Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg Thr Gln Glu Ser 195 200 205 gaa tgt gtt tgt atc aat ggg act tgt aca gta gta atg act gat gga 672 Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly 210 215 220 agt gct tca gga aga gct gat act aaa ata cta ttc att gaa gag ggg 720 Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly 225 230 235 240 aaa att gtt cat att agc cca ttg tca gga agt gct cag cat gta gag 768 Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gln His Val Glu 245 250 255 gag tgt tcc tgt tat cct cga tat cct ggt gtc aga tgt atc tgc aga 816 Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Ile Cys Arg 260 265 270 gac aac tgg aaa ggc tct aat agg cca gtc gta gat ata aat gtg aaa 864 Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp Ile Asn Val Lys 275 280 285 gat tat agc att gat tcc agt tat gtg tgc tca ggg ctt gtt ggc gac 912 Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp 290 295 300 aca ccc aga aaa aac gac aga tct agc agt agc tat tgc cgg aat cct 960 Thr Pro Arg Lys Asn Asp Arg Ser Ser Ser Ser Tyr Cys Arg Asn Pro 305 310 315 320 aac aat gaa aaa ggg aat cac gga gtg aaa ggc tgg gcc ttt gac gat 1008 Asn Asn Glu Lys Gly Asn His Gly Val Lys Gly Trp Ala Phe Asp Asp 325 330 335 gga aat gac gtg tgg atg gga aga acg atc agc gag gat tca cgc tca 1056 Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Asp Ser Arg Ser 340 345 350 ggt tat gaa acc ttc aaa gtc att ggt ggt tgg tcc aca cct aat tcc 1104 Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser Thr Pro Asn Ser 355 360 365 aaa ttg cag ata aat agg caa gtc ata gtt gac agc gct aat agg tca 1152 Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Ser Ala Asn Arg Ser 370 375 380 ggt tat tct ggt att ttc tct gtt gag ggc aaa agc tgc atc aat agg 1200 Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg 385 390 395 400 tgc ttt tat gtg gag ttg ata agg gga agg gaa cag gaa act aga gta 1248 Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Glu Gln Glu Thr Arg Val 405 410 415 tgg tgg acc tca aac agt att gtt gtg ttt tgt ggc act tca ggt acc 1296 Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr 420 425 430 tat ggg aca ggc tca tgg cct gat ggg gcg gac atc aat ctc atg cct 1344 Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu Met Pro 435 440 445 ata taa 1350 Ile 2 449 PRT Artificial Sequence part of pACGCN4Nas 2 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Gln Asp Leu Gly Gly Met Lys Gln Ile Glu Asp Lys Leu Glu Glu Ile 20 25 30 Leu Ser Lys Leu Tyr His Ile Glu Asn Glu Leu Ala Arg Ile Lys Lys 35 40 45 Leu Leu Gly Glu Gly Gly Lys Glu Ile Cys Pro Lys Leu Val Glu Tyr 50 55 60 Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly Phe Ala Pro Phe 65 70 75 80 Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val 85 90 95 Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys Cys Tyr Gln Phe 100 105 110 Ala Leu Gly Gln Gly Thr Thr Leu Glu Asn Lys His Ser Asn Asp Thr 115 120 125 Ile His Asp Arg Thr Pro His Arg Thr Leu Leu Met Asn Glu Leu Gly 130 135 140 Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile Ala Trp Ser Ser 145 150 155 160 Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly 165 170 175 Tyr Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val 180 185 190 Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg Thr Gln Glu Ser 195 200 205 Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly 210 215 220 Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly 225 230 235 240 Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gln His Val Glu 245 250 255 Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Ile Cys Arg 260 265 270 Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp Ile Asn Val Lys 275 280 285 Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp 290 295 300 Thr Pro Arg Lys Asn Asp Arg Ser Ser Ser Ser Tyr Cys Arg Asn Pro 305 310 315 320 Asn Asn Glu Lys Gly Asn His Gly Val Lys Gly Trp Ala Phe Asp Asp 325 330 335 Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Asp Ser Arg Ser 340 345 350 Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser Thr Pro Asn Ser 355 360 365 Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Ser Ala Asn Arg Ser 370 375 380 Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg 385 390 395 400 Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Glu Gln Glu Thr Arg Val 405 410 415 Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr 420 425 430 Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu Met Pro 435 440 445 Ile 3 282 DNA Artificial Sequence part of pACsM2eGCN4 3 atg cta cta gta aat cag tca cac caa ggc ttc aat aag gaa cac aca 48 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 agc aag atg gta agc gct att gtt tta tat gtg ctt ttg gcg gcg gcg 96 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 gcg cat tct gcc ttt gcc tct ctg ctg acc gaa gtt gaa acc cct atc 144 Ala His Ser Ala Phe Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Ile 35 40 45 aga aac gaa tgg ggg tgc aga tgc aac gat tca tcc gga ggt atg aaa 192 Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Gly Gly Met Lys 50 55 60 caa atc gaa gac aag ctg gaa gaa atc ctt tcg aaa ctg tac cac atc 240 Gln Ile Glu Asp Lys Leu Glu Glu Ile Leu Ser Lys Leu Tyr His Ile 65 70 75 80 gaa aac gag ctg gcc agg atc aag aaa ctg ctg ggc gaa tag 282 Glu Asn Glu Leu Ala Arg Ile Lys Lys Leu Leu Gly Glu 85 90 4 93 PRT Artificial Sequence part of pACsM2eGCN4 4 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 Ala His Ser Ala Phe Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Ile 35 40 45 Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Gly Gly Met Lys 50 55 60 Gln Ile Glu Asp Lys Leu Glu Glu Ile Leu Ser Lys Leu Tyr His Ile 65 70 75 80 Glu Asn Glu Leu Ala Arg Ile Lys Lys Leu Leu Gly Glu 85 90 5 1230 DNA Artificial Sequence part of pACsM2eGCN4C3d 5 atg cta cta gta aat cag tca cac caa ggc ttc aat aag gaa cac aca 48 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 agc aag atg gta agc gct att gtt tta tat gtg ctt ttg gcg gcg gcg 96 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 gcg cat tct gcc ttt gcc tct ctg ctg acc gaa gtt gaa acc cct atc 144 Ala His Ser Ala Phe Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Ile 35 40 45 aga aac gaa tgg ggg tgc aga tgc aac gat tca tcc gga ggt atg aaa 192 Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Gly Gly Met Lys 50 55 60 caa atc gaa gac aag ctg gaa gaa atc ctt tcg aaa ctg tac cac atc 240 Gln Ile Glu Asp Lys Leu Glu Glu Ile Leu Ser Lys Leu Tyr His Ile 65 70 75 80 gaa aac gag ctg gcc agg atc aag aaa ctg ctg ggc gaa gga tca ggt 288 Glu Asn Glu Leu Ala Arg Ile Lys Lys Leu Leu Gly Glu Gly Ser Gly 85 90 95 tca ccg gat ctc gga tct acc ccc gca ggc tct ggg gaa cag aac atg 336 Ser Pro Asp Leu Gly Ser Thr Pro Ala Gly Ser Gly Glu Gln Asn Met 100 105 110 att ggc atg aca cca aca gtc att gcg gta cac tac ctg gac cag acc 384 Ile Gly Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln Thr 115 120 125 gaa cag tgg gag aag ttc ggc ata gag aag agg caa gag gcc ctg gag 432 Glu Gln Trp Glu Lys Phe Gly Ile Glu Lys Arg Gln Glu Ala Leu Glu 130 135 140 ctc atc aag aaa ggg tac acc cag cag ctg gcc ttc aaa cag ccc agc 480 Leu Ile Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Lys Gln Pro Ser 145 150 155 160 tct gcc tat gct gcc ttc aac aac cgg ccc ccc agc acc tgg ctg aca 528 Ser Ala Tyr Ala Ala Phe Asn Asn Arg Pro Pro Ser Thr Trp Leu Thr 165 170 175 gcc tac gtg gtc aag gtc ttc tct cta gct gcc aac ctc atc gcc atc 576 Ala Tyr Val Val Lys Val Phe Ser Leu Ala Ala Asn Leu Ile Ala Ile 180 185 190 gac tct cac gtc ctg tgt ggg gct gtt aaa tgg ttg att ctg gag aaa 624 Asp Ser His Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys 195 200 205 cag aag ccg gat ggt gtc ttt cag gag gat ggg ccc gtg att cac caa 672 Gln Lys Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His Gln 210 215 220 gaa atg att ggt ggc ttc cgg aac gcc aag gag gca gat gtg tca ctc 720 Glu Met Ile Gly Gly Phe Arg Asn Ala Lys Glu Ala Asp Val Ser Leu 225 230 235 240 aca gcc ttc gtc ctc atc gca ctg cag gaa gcc agg gac atc tgt gag 768 Thr Ala Phe Val Leu Ile Ala Leu Gln Glu Ala Arg Asp Ile Cys Glu 245 250 255 ggg cag gtc aat agc ctt cct ggg agc atc aac aag gca ggg gag tat 816 Gly Gln Val Asn Ser Leu Pro Gly Ser Ile Asn Lys Ala Gly Glu Tyr 260 265 270 att gaa gcc agt tac atg aac ctg cag aga cca tac aca gtg gcc att 864 Ile Glu Ala Ser Tyr Met Asn Leu Gln Arg Pro Tyr Thr Val Ala Ile 275 280 285 gct ggg tat gcc ctg gcc ctg atg aac aaa ctg gag gaa cct tac ctc 912 Ala Gly Tyr Ala Leu Ala Leu Met Asn Lys Leu Glu Glu Pro Tyr Leu 290 295 300 ggc aag ttt ctg aac aca gcc aaa gat cgg aac cgc tgg gag gag cct 960 Gly Lys Phe Leu Asn Thr Ala Lys Asp Arg Asn Arg Trp Glu Glu Pro 305 310 315 320 gac cag cag ctc tac aac gta gag gcc aca tcc tac gcc ctc ctg gcc 1008 Asp Gln Gln Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala 325 330 335 ctg ctg ctg ctg aaa gac ttt gac tct gtg ccc cct gta gtg cgc tgg 1056 Leu Leu Leu Leu Lys Asp Phe Asp Ser Val Pro Pro Val Val Arg Trp 340 345 350 ctc aat gag caa aga tac tac gga ggc ggc tat ggc tcc acc cag gct 1104 Leu Asn Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala 355 360 365 acc ttc atg gta ttc caa gcc ttg gcc caa tat caa aca gat gtc cct 1152 Thr Phe Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Thr Asp Val Pro 370 375 380 gac cat aag gac ttg aac atg gat gtg tcc ttc cac ctc ccc agc agt 1200 Asp His Lys Asp Leu Asn Met Asp Val Ser Phe His Leu Pro Ser Ser 385 390 395 400 gga tct gaa gag ttc gga tcg gat cct tga 1230 Gly Ser Glu Glu Phe Gly Ser Asp Pro 405 6 409 PRT Artificial Sequence part of pACsM2eGCN4C3d 6 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 Ala His Ser Ala Phe Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Ile 35 40 45 Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Gly Gly Met Lys 50 55 60 Gln Ile Glu Asp Lys Leu Glu Glu Ile Leu Ser Lys Leu Tyr His Ile 65 70 75 80 Glu Asn Glu Leu Ala Arg Ile Lys Lys Leu Leu Gly Glu Gly Ser Gly 85 90 95 Ser Pro Asp Leu Gly Ser Thr Pro Ala Gly Ser Gly Glu Gln Asn Met 100 105 110 Ile Gly Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln Thr 115 120 125 Glu Gln Trp Glu Lys Phe Gly Ile Glu Lys Arg Gln Glu Ala Leu Glu 130 135 140 Leu Ile Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Lys Gln Pro Ser 145 150 155 160 Ser Ala Tyr Ala Ala Phe Asn Asn Arg Pro Pro Ser Thr Trp Leu Thr 165 170 175 Ala Tyr Val Val Lys Val Phe Ser Leu Ala Ala Asn Leu Ile Ala Ile 180 185 190 Asp Ser His Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys 195 200 205 Gln Lys Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His Gln 210 215 220 Glu Met Ile Gly Gly Phe Arg Asn Ala Lys Glu Ala Asp Val Ser Leu 225 230 235 240 Thr Ala Phe Val Leu Ile Ala Leu Gln Glu Ala Arg Asp Ile Cys Glu 245 250 255 Gly Gln Val Asn Ser Leu Pro Gly Ser Ile Asn Lys Ala Gly Glu Tyr 260 265 270 Ile Glu Ala Ser Tyr Met Asn Leu Gln Arg Pro Tyr Thr Val Ala Ile 275 280 285 Ala Gly Tyr Ala Leu Ala Leu Met Asn Lys Leu Glu Glu Pro Tyr Leu 290 295 300 Gly Lys Phe Leu Asn Thr Ala Lys Asp Arg Asn Arg Trp Glu Glu Pro 305 310 315 320 Asp Gln Gln Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala 325 330 335 Leu Leu Leu Leu Lys Asp Phe Asp Ser Val Pro Pro Val Val Arg Trp 340 345 350 Leu Asn Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala 355 360 365 Thr Phe Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Thr Asp Val Pro 370 375 380 Asp His Lys Asp Leu Asn Met Asp Val Ser Phe His Leu Pro Ser Ser 385 390 395 400 Gly Ser Glu Glu Phe Gly Ser Asp Pro 405 7 24 DNA Artificial Sequence primer BACfor 7 tttactgttt tcgtaacagt tttg 24 8 54 DNA Artificial Sequence primer GCN4nh 8 tacagaagct tgtcttcgat ttgtttcata ccgccaaggt cttgggcgaa aacc 54 9 54 DNA Artificial Sequence primer GCN4cooh 9 atctgatcaa gaaactgctg ggcgaaggtg gcaaagagat atgccccaaa ttag 54 10 23 DNA Artificial Sequence primer BACrev 10 cattttatgt ttcaggttca ggg 23 11 25 DNA Artificial Sequence primer GP67s 11 gctactagta aatcagtcac accaa 25 12 33 DNA Artificial Sequence primer GP67a 12 cgaagcttgc cggcaaaggc agaatgcgcc gcc 33 13 30 DNA Artificial Sequence primer M2rev 13 accattccgg atgaatcgtt gcatctgcac 30 14 23 DNA Artificial Sequence primer M2Ss 14 tctctgctga ccgaagttga aac 23 15 50 DNA Artificial Sequence primer UM2ECa 15 cgaagcttac tagttcacgg atccccactt gaatcgttgc atctgcaccc 50 16 32 DNA Artificial Sequence primer GCN4for 16 agatttccgg aggtatgaaa caaatcgaag ac 32 17 31 DNA Artificial Sequence primer GCN4rev 17 ataggagatc tattcgccca gcagtttctt g 31 18 43 DNA Artificial Sequence primer GCNrev2 18 tattggatcc ggtgaacctg atccttcgcc cagcagtttc ttg 43 19 52 DNA Artificial Sequence primer GCN4pos 19 agctggaaga aatcctttcg aaactgtacc acatcgaaaa cgagctggcc ag 52 20 52 DNA Artificial Sequence primer GCN4neg 20 gatcctggcc agctcgtttt cgatgtggta cagtttcgaa aggatttctt cc 52 21 35 DNA Artificial Sequence primer C3ds 21 ccgcgcccac ccgacgagat ctcggatcta ccccc 35 22 38 DNA Artificial Sequence primer C3da 22 gcactagttc aaggatccga tccgaactct tcagatcc 38 

1. A chimeric protein comprising an antigen, derived from a naturally occurring oligomeric protein complex, and an oligomerisation domain.
 2. The chimeric protein according to claim 1 whereby said antigen is an influenza antigen.
 3. The chimeric protein according to claim 2 whereby said antigen is influenza neuraminidase or a functional fragment thereof.
 4. The chimeric protein according to claim 2 whereby said antigen is influenza A M2 or a functional fragment thereof.
 5. The chimeric protein according to claim 2 whereby said antigen is influenza B NB or a functional fragment thereof.
 6. The chimeric protein according to any of the claims 1-5 whereby said oligomerisation domain is a leucine zipper.
 7. A recombinant oligomeric protein complex comprising a chimeric protein according to any of the claims 1-6.
 8. The recombinant oligomeric protein complex according to claim 7 whereby said recombinant oligomeric protein complex has a comparable enzymatic activity as the naturally occurring ogliomeric protein complex, of which the antigen of the chimeric protein is derived.
 9. The recombinant oligomeric protein complex according to claim 7 or 8 whereby said recombinant oligomeric protein complex is a tetramer.
 10. The recombinant oligomeric protein complex according to claim 7 or 8 whereby said recombinant oligomeric protein complex is a dimer.
 11. A nucleic acid encoding a chimeric protein according to any of the claims 1-6.
 12. The nucleic acid according to claim 11, comprising the sequence presented in SEQ ID N°1.
 13. The nucleic acid according to claim 11, comprising the sequence presented in SEQ ID N°3.
 14. The nucleic acid according to claim 11, comprising the sequence presented in SEQ ID N°5.
 15. An expression vector, comprising a nucleic acid according to any of the claims 11-14.
 16. A host cell, comprising an expression vector according to claim
 15. 17. Use of a chimeric protein according to any of the claims 1-6 and/or a recombinant oligomeric protein complex according to any of the claims 7-10 for the preparation of a vaccine.
 18. Use of a chimeric protein and/or a recombinant oligomeric protein complex according to claim 17, whereby the vaccine is an influenza vaccine.
 19. Use of a chimeric protein according to claim 18 whereby said chimeric protein comprises SEQ ID N°2, SEQ ID N°4 or SEQ ID N°6.
 20. Vaccine against influenza, comprising a chimeric protein according to any of the claims 1-6 and/or a recombinant oligomeric protein complex according to any of the claims 7-10.
 21. Vaccine against influenza, comprising a substantially pure tetrameric form of a recombinant influenza antigen.
 22. Vaccine according to claim 21, whereby the antigen is influenza neuraminidase or a functional fragment thereof.
 23. Vaccine according to claim 21, whereby the antigen is influenza A M2 or a functional fragment thereof.
 24. Vaccine according to claim 21, whereby the antigen is influenza B NB or a functional fragment thereof.
 25. Vaccine according to claim 22, whereby said substantially pure tetrameric form has an enzymatic activity that is comparable to naturally occurring influenza neuraminidase.
 26. Vaccine against influenza comprising a nucleic acid according to claims 11-14. 